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A micro-technique for detection of leukocyte agglutinins
Journal oflmmunologicalMethods 7 (1975) 103-108
© North-Holland Publishing Company
A MICRO-TECHNIQUE FOR DETECTION OF LEUKOCYTE AGGLUTININS* An-Fu JIANG and Parviz LALEZARI The Division o f lmmunohematology, Department o f Medicine, Montefiore tlospital & Medical Center, and Albert Einstein College o f Medicine, New York, N. Y., U.S.A.
Received 27 August 1974,
accepted 18 October 1974
A method which uses microquantities of reagents is described for detection of agglutinating leukocyte antibodies. The leukocyte preparations are devoid of platelets, and have little red cell contamination. Both serum and plasma can be used as the source of antibody. Leukocyte antibodies, particularly those specific for neutrophils, are detected by this method.
1. Introduction Granulocyte fragility and their tendency to spontaneously aggregate and disintegrate have been major factors in complicating the leukocyte agglutination test, as it was originally described by Moeschlin and Wagner (1952) and by Dausset and Nenna (1952). These methods, which employed heparinized or defibrinated blood, were subsequently modified by the introduction of the anticoagulant ethylenediamine tetra-acetate (EDTA)(Wasastjerna, 1954; Lalezari et al., 1960). This chelating agent was shown to preserve the granulocytes from rapid disintegration and non-specific aggregation, as well as to inhibit the l e u k o c y t e - r e d cell mixed agglutination reaction due to red cell ABO incompatibility (Lalezari and Spaet, 1959). The presence of EDTA in the reaction mixture was also found to facilitate the demonstration of certain anti-leukocyte antibodies, particularly those specific for blood neutrophils (Lalezari and Bernard, 1966). More recently, Cruikshank and Hay (1973) have described a microagglutination test in which papain is used to enhance the reaction. We would like to report an EDTA-dependent microagglutination technique which provides a high degree of reproducibility, and a sensitivity equivalent to the macroagglutination test previously described (Lalezari and Bernard, 1964).
* Supported by National Institutes of Health Contract 69-2071 and USPttS Grant 4&TA1531801A1HEM. 103
104
A-bl Jiang, P. Lalezari, Micro-technique Jbr leukoagglutinins
2. Materials and methods
2.1. Reagents Anti-H lectin was prepared as a saline extract from the seeds of Ulex europeaus. Tire final extract had a minimum titer of 64 on immediate centrifugation with human group O red cells. Anti-A,B, prepared from immunized group O blood donors (Ortho Diagnostics, Raritan, New Jersey), had a minimum titer of 512 against A and 128 against B red cells, and was shown to contain no leukoagglutinins. Hexadimethrine bromide (Polybrene ®, Aldrich Chemical Co., Milwaukee, Wisconsin) was used as a 1% solution in 0.9% NaC1. The leukocyte wash solution contained 1 volume normal serum, 9 volumes 0.9% NaC1, and 0.5 volumes 10% disodium-EDTA. EDTA-serum, for the suspension of leukocytes, was prepared by the addition of 0.7 ml of EDTA to 10 ml of heat-inactivated normal serum.
2.2. Preparation o f leukocTtes The basic method for cell preparation has been previously reported (Lalezari et al., 1970). It is based on the finding that the hemagglutination produced by either anti-H lectin or by anti-A,B antibody is synergistic with the hemagglutination produced by hexadimethrine bromide (Polybrene ®) (Lalezari and Spaet, 1961). The combined use of these agents causes rapid and selective sedimentation of the red blood cells. Blood is defibrinated by continuous mixing of the freshly drawn blood in a flask which contains one 2 mm glass bead for each ml of blood. Following defibrination and removal of the fibrin platelet clot, 0.05 ml of 10% EDTA is added to each ml of blood. A 1 ml aliquot is then placed in a plastic tube and centrifuged at 3 5 0 0 g for 90 sec in a Model 52 Fisher centrifuge. The supernatant serum is used for preparation of leukocyte-wash solution or is discarded. The buff:/ coat is removed by a Pasteur pipette and transferred to a disposable micro-beaker (Cat. V9330, 5 ml, Aloe Scientific, St. Louis, Mo.). This beaker contains 0.4 ml leukocyte-wash solution and 0.3 ml of Ulex extract for group O blood. When group A or B blood is used, 0.3 ml of anti-A,B is used instead of Ulex extract; tire addition of 0.2 ml of lectin further potentiates the red cell agglutination produced by anti-A,B. The beaker is placed on a shaker (Fisher Rotator, Fisher Scientific, Edison, New Jersey) for 3 rain; one drop of Polybrene ¢~ is added and shaking is continued for an additional 2 min. At the end of this time, large red cell aggregates are formed. The mixture is poured into a Fisher centrifuge tube and allowed to sediment for 1 min, after wlzich time tire tube is centrifuged at 500 g for 20 sec. The leukocyte-rich supernatant is then transferred to another tube and recentrifuged for 15 sec at 3 5 0 0 g . This centrifugation sediments the leukocytes and also facilitates clumping of the remaining, non-agglutinated red cells. Gentle resuspension of the cells with the aid of a Pasteur pipette, followed by 15 sec centrifugation at 500 g would eliminate most of tire red cell aggregates. Repeating this step may be necessary when O type red cells are used.
A-F. Jiang. P. Lalezari, Micro-technique for leukoagglutinins
105
Following a 15 sec centrifugation at 3500 g, the supernatant fluid is replaced by the wash solution. The cell button is again redispersed with a Pasteur pipette, and this allows resuspension and washing of the leukocytes, but the residual red cells remain aggregated. These aggregates are sedimented by 15 sec centrifugation at 500 g. The leukocyte-rich supernatant is transferred into another Fisher tube and centrifuged at 3500 g for 10 sec. The supernatant is discarded and again replaced by the leukocyte wash solution and the cells are resuspended. In the next step, the cells are washed with 3% BSA. For this purpose, the ceils are centrifuged at 3500 g for 10 sec, the supernatant is replaced by 3% BSA and the cells are mixed. The suspension is again centrifuged for 10 sec at 3500 g. The BSA supernatant is replaced by the EDTA-serum, the cells are resuspended, and tile count is adjusted to 5000/mm 3. This cell preparation can then be kept at room temperature for several hours before using. 2.3. Preparatkm o f trays and the test procedure
Falcon trays* (Falcon Plastics, Oxnard, Calif.) are first flooded with mineral oil and then tilted to remove excess oil, leaving the chambers covered by a thin oil fihn. Serial 2-fold dilutions of the test plasmas (or sera) are prepared in 0.9% NaCI. Using a Hamilton syringe and dispenser, six X from each dilution are placed under oil in each chamber. Controls consist of normal plasma (or serum) and its dilutions dispensed in the same manner. One X of 1% Na3N is added to the undiluted test and control plasmas if a prolonged incubation is anticipated. It has been found, experimentally, that the incubated granulocytes are better preserved if bacterial growth is reduced by the addition of Na3N. The test is performed by adding one of the cell suspension to each chamber and incubating the trays at 37°C. Placing the trays on a rotator expedites the reaction. The results may be evaluated after 2 and 5 hr. The trays are examined under an inverted phase microscope, and the strength of the reaction is arbitrarily graded from 0 4, according to the size of the aggregates and proportion of the cells participating in the reaction. Examples of positive and negative reactions are shown in fig. 1. Table 1 shows the results obtained after 5 hr incubation at 37°C by the present method as compared with those obtained by the previously described method after 18 hr incubation (Lalezari and Bernard, 1964). It may be observed that there is a complete agreement between the two tests, and a similar level of sensitivity for both procedures.
3. Discussion The procedure described here provides a method for detection of leukocyte antibodies, particularly those specific for neutrophils (table 1). Both serum and * Cat. #3034 MicroTest.
* Specificity not established be performed.
by the present
method,
supply
0
20 48 19 168 31 512
0
10 36 43 19 23 7
of antisera,
the macro-technique
10 36 43 19 23 7 5 9 6 6
0
No. positive
titer
Microagglutination Average
Macroagglutination No. positive
and due to inadequate
NAl NAl NA2 NBl ‘Vaz” 5b (9 a*1 (4 a*1 (4 b*1 (4 c*1
58 15 49 58 22 24 I 10 9 10 10
Normal DeR. v.d.B. CR. Parker Vaz Ke Gerritsen-Bakker Hekkens (NIH) Oshita (NIH) S.A. (NIH)
(NIII)
Specificity
tests
No. tested
agglutination
Serum
Table 1 Results of leukocyte
Average
could not
24 64 46 165 12 512 48 22 28 15
0
titer
A-k: Jiang, P. Lalezari, Micro-technique Jbr leukoagglutinins
l:ig. 1: A
positive agglutination reaction; B
negative control.
plasma, in microquantities, may be used as the source of antibody. The cell preparation contains granulocytes, mononuclear cells, and the red cell contamination is as low as 1%, platelets being absent. When blood type O is used, red cell contamination may exceed this ratio. This is due to the inefficiency of lectin as compared to the anti-A,B antibody in agglutinating red cells. This red cell contamination may be reduced by repeating the centrifugation steps in the presence of lectin and Polybrene o. The minimum proportion of tire neutrophilic cells in the cell preparation required for neutrophil antibody detection remains to be determined. Obviously, the ratio of neutrophils to other nucleated cells in the preparation would depend upon their ratio in the original blood sanrple; however, it has previously been shown that the use of Polybrene causes a selective reduction in the ratio of lymphocytes (Lalezari, 1962). The results of the test should be read after two and again after 5 hr of 37 °C incubation. Since prolonged incubation at 37°C may cause cell disintegration in the chambers containing undiluted sera, it would be desirable to examine the trays after 2 hr of incubation, for evaluation of the results in the chambers which contain undilute sera. The end-point then can be determined after 5 hr of incubation. Cell alteration is minimum if trays are kept at 24°C, even for as long as 24 hr. Reactivity of certain antibodies, such as anti-NA2, has been found to increase by this prolonged room temperature incubation, especially if the trays are placed on a shaker. The present method has been successfully used to type blood neutrophils, and the microquantity required has been especially valuable in assaying various soluble fractions of neutrophils for their antigenic activity (P. Lalezari and A.F. Jiang, unpublished observations).
108
A -F. Jiang, P. Lalezari, Micro-technique for leukoagglutinins
References Cruikshank, J.A. and J. tlay, 1973, Tissue Antigens 3, 57. Dausset, J.and A. Nenna, 1952, C.R. Soc. Biol. 146, 1539. Lalezari, P., 1962, Blood 19,109. Lalezari, P. and G.E. Bernard, 1964, Vo×. Sanguinis 9,644. Lalezari, P. and G.E. Bernard, 1966, J. Clin. Invest. 45, 1741. Lalezari, P., M. Nussbaum, S. Gelman and T.H. Spaet, 1960, Blood 15,236. Lalezari, P. and T.H. Spaet, 1959, Federation Proc. 18, 85. Lalezari, P. and T.H. Spaet, 1961, J. Lab. Clin. Meal. 57,868. Lalezari, P., W.J., Weinstein and B. Thalenfeld, 1970, in Terasaki, P.I. (ed.): Histocompatibilit} Testing, Munksgaard, Copenhagen, p. 319. Moeschlin, S. and K. Wagner, 1952, Schweiz. Med. Wochenschr. 82, 1104. Wasastjerna, C., Acta Med. Scan. 149,355.
© North-Holland Publishing Company
A MICRO-TECHNIQUE FOR DETECTION OF LEUKOCYTE AGGLUTININS* An-Fu JIANG and Parviz LALEZARI The Division o f lmmunohematology, Department o f Medicine, Montefiore tlospital & Medical Center, and Albert Einstein College o f Medicine, New York, N. Y., U.S.A.
Received 27 August 1974,
accepted 18 October 1974
A method which uses microquantities of reagents is described for detection of agglutinating leukocyte antibodies. The leukocyte preparations are devoid of platelets, and have little red cell contamination. Both serum and plasma can be used as the source of antibody. Leukocyte antibodies, particularly those specific for neutrophils, are detected by this method.
1. Introduction Granulocyte fragility and their tendency to spontaneously aggregate and disintegrate have been major factors in complicating the leukocyte agglutination test, as it was originally described by Moeschlin and Wagner (1952) and by Dausset and Nenna (1952). These methods, which employed heparinized or defibrinated blood, were subsequently modified by the introduction of the anticoagulant ethylenediamine tetra-acetate (EDTA)(Wasastjerna, 1954; Lalezari et al., 1960). This chelating agent was shown to preserve the granulocytes from rapid disintegration and non-specific aggregation, as well as to inhibit the l e u k o c y t e - r e d cell mixed agglutination reaction due to red cell ABO incompatibility (Lalezari and Spaet, 1959). The presence of EDTA in the reaction mixture was also found to facilitate the demonstration of certain anti-leukocyte antibodies, particularly those specific for blood neutrophils (Lalezari and Bernard, 1966). More recently, Cruikshank and Hay (1973) have described a microagglutination test in which papain is used to enhance the reaction. We would like to report an EDTA-dependent microagglutination technique which provides a high degree of reproducibility, and a sensitivity equivalent to the macroagglutination test previously described (Lalezari and Bernard, 1964).
* Supported by National Institutes of Health Contract 69-2071 and USPttS Grant 4&TA1531801A1HEM. 103
104
A-bl Jiang, P. Lalezari, Micro-technique Jbr leukoagglutinins
2. Materials and methods
2.1. Reagents Anti-H lectin was prepared as a saline extract from the seeds of Ulex europeaus. Tire final extract had a minimum titer of 64 on immediate centrifugation with human group O red cells. Anti-A,B, prepared from immunized group O blood donors (Ortho Diagnostics, Raritan, New Jersey), had a minimum titer of 512 against A and 128 against B red cells, and was shown to contain no leukoagglutinins. Hexadimethrine bromide (Polybrene ®, Aldrich Chemical Co., Milwaukee, Wisconsin) was used as a 1% solution in 0.9% NaC1. The leukocyte wash solution contained 1 volume normal serum, 9 volumes 0.9% NaC1, and 0.5 volumes 10% disodium-EDTA. EDTA-serum, for the suspension of leukocytes, was prepared by the addition of 0.7 ml of EDTA to 10 ml of heat-inactivated normal serum.
2.2. Preparation o f leukocTtes The basic method for cell preparation has been previously reported (Lalezari et al., 1970). It is based on the finding that the hemagglutination produced by either anti-H lectin or by anti-A,B antibody is synergistic with the hemagglutination produced by hexadimethrine bromide (Polybrene ®) (Lalezari and Spaet, 1961). The combined use of these agents causes rapid and selective sedimentation of the red blood cells. Blood is defibrinated by continuous mixing of the freshly drawn blood in a flask which contains one 2 mm glass bead for each ml of blood. Following defibrination and removal of the fibrin platelet clot, 0.05 ml of 10% EDTA is added to each ml of blood. A 1 ml aliquot is then placed in a plastic tube and centrifuged at 3 5 0 0 g for 90 sec in a Model 52 Fisher centrifuge. The supernatant serum is used for preparation of leukocyte-wash solution or is discarded. The buff:/ coat is removed by a Pasteur pipette and transferred to a disposable micro-beaker (Cat. V9330, 5 ml, Aloe Scientific, St. Louis, Mo.). This beaker contains 0.4 ml leukocyte-wash solution and 0.3 ml of Ulex extract for group O blood. When group A or B blood is used, 0.3 ml of anti-A,B is used instead of Ulex extract; tire addition of 0.2 ml of lectin further potentiates the red cell agglutination produced by anti-A,B. The beaker is placed on a shaker (Fisher Rotator, Fisher Scientific, Edison, New Jersey) for 3 rain; one drop of Polybrene ¢~ is added and shaking is continued for an additional 2 min. At the end of this time, large red cell aggregates are formed. The mixture is poured into a Fisher centrifuge tube and allowed to sediment for 1 min, after wlzich time tire tube is centrifuged at 500 g for 20 sec. The leukocyte-rich supernatant is then transferred to another tube and recentrifuged for 15 sec at 3 5 0 0 g . This centrifugation sediments the leukocytes and also facilitates clumping of the remaining, non-agglutinated red cells. Gentle resuspension of the cells with the aid of a Pasteur pipette, followed by 15 sec centrifugation at 500 g would eliminate most of tire red cell aggregates. Repeating this step may be necessary when O type red cells are used.
A-F. Jiang. P. Lalezari, Micro-technique for leukoagglutinins
105
Following a 15 sec centrifugation at 3500 g, the supernatant fluid is replaced by the wash solution. The cell button is again redispersed with a Pasteur pipette, and this allows resuspension and washing of the leukocytes, but the residual red cells remain aggregated. These aggregates are sedimented by 15 sec centrifugation at 500 g. The leukocyte-rich supernatant is transferred into another Fisher tube and centrifuged at 3500 g for 10 sec. The supernatant is discarded and again replaced by the leukocyte wash solution and the cells are resuspended. In the next step, the cells are washed with 3% BSA. For this purpose, the ceils are centrifuged at 3500 g for 10 sec, the supernatant is replaced by 3% BSA and the cells are mixed. The suspension is again centrifuged for 10 sec at 3500 g. The BSA supernatant is replaced by the EDTA-serum, the cells are resuspended, and tile count is adjusted to 5000/mm 3. This cell preparation can then be kept at room temperature for several hours before using. 2.3. Preparatkm o f trays and the test procedure
Falcon trays* (Falcon Plastics, Oxnard, Calif.) are first flooded with mineral oil and then tilted to remove excess oil, leaving the chambers covered by a thin oil fihn. Serial 2-fold dilutions of the test plasmas (or sera) are prepared in 0.9% NaCI. Using a Hamilton syringe and dispenser, six X from each dilution are placed under oil in each chamber. Controls consist of normal plasma (or serum) and its dilutions dispensed in the same manner. One X of 1% Na3N is added to the undiluted test and control plasmas if a prolonged incubation is anticipated. It has been found, experimentally, that the incubated granulocytes are better preserved if bacterial growth is reduced by the addition of Na3N. The test is performed by adding one of the cell suspension to each chamber and incubating the trays at 37°C. Placing the trays on a rotator expedites the reaction. The results may be evaluated after 2 and 5 hr. The trays are examined under an inverted phase microscope, and the strength of the reaction is arbitrarily graded from 0 4, according to the size of the aggregates and proportion of the cells participating in the reaction. Examples of positive and negative reactions are shown in fig. 1. Table 1 shows the results obtained after 5 hr incubation at 37°C by the present method as compared with those obtained by the previously described method after 18 hr incubation (Lalezari and Bernard, 1964). It may be observed that there is a complete agreement between the two tests, and a similar level of sensitivity for both procedures.
3. Discussion The procedure described here provides a method for detection of leukocyte antibodies, particularly those specific for neutrophils (table 1). Both serum and * Cat. #3034 MicroTest.
* Specificity not established be performed.
by the present
method,
supply
0
20 48 19 168 31 512
0
10 36 43 19 23 7
of antisera,
the macro-technique
10 36 43 19 23 7 5 9 6 6
0
No. positive
titer
Microagglutination Average
Macroagglutination No. positive
and due to inadequate
NAl NAl NA2 NBl ‘Vaz” 5b (9 a*1 (4 a*1 (4 b*1 (4 c*1
58 15 49 58 22 24 I 10 9 10 10
Normal DeR. v.d.B. CR. Parker Vaz Ke Gerritsen-Bakker Hekkens (NIH) Oshita (NIH) S.A. (NIH)
(NIII)
Specificity
tests
No. tested
agglutination
Serum
Table 1 Results of leukocyte
Average
could not
24 64 46 165 12 512 48 22 28 15
0
titer
A-k: Jiang, P. Lalezari, Micro-technique Jbr leukoagglutinins
l:ig. 1: A
positive agglutination reaction; B
negative control.
plasma, in microquantities, may be used as the source of antibody. The cell preparation contains granulocytes, mononuclear cells, and the red cell contamination is as low as 1%, platelets being absent. When blood type O is used, red cell contamination may exceed this ratio. This is due to the inefficiency of lectin as compared to the anti-A,B antibody in agglutinating red cells. This red cell contamination may be reduced by repeating the centrifugation steps in the presence of lectin and Polybrene o. The minimum proportion of tire neutrophilic cells in the cell preparation required for neutrophil antibody detection remains to be determined. Obviously, the ratio of neutrophils to other nucleated cells in the preparation would depend upon their ratio in the original blood sanrple; however, it has previously been shown that the use of Polybrene causes a selective reduction in the ratio of lymphocytes (Lalezari, 1962). The results of the test should be read after two and again after 5 hr of 37 °C incubation. Since prolonged incubation at 37°C may cause cell disintegration in the chambers containing undiluted sera, it would be desirable to examine the trays after 2 hr of incubation, for evaluation of the results in the chambers which contain undilute sera. The end-point then can be determined after 5 hr of incubation. Cell alteration is minimum if trays are kept at 24°C, even for as long as 24 hr. Reactivity of certain antibodies, such as anti-NA2, has been found to increase by this prolonged room temperature incubation, especially if the trays are placed on a shaker. The present method has been successfully used to type blood neutrophils, and the microquantity required has been especially valuable in assaying various soluble fractions of neutrophils for their antigenic activity (P. Lalezari and A.F. Jiang, unpublished observations).
108
A -F. Jiang, P. Lalezari, Micro-technique for leukoagglutinins
References Cruikshank, J.A. and J. tlay, 1973, Tissue Antigens 3, 57. Dausset, J.and A. Nenna, 1952, C.R. Soc. Biol. 146, 1539. Lalezari, P., 1962, Blood 19,109. Lalezari, P. and G.E. Bernard, 1964, Vo×. Sanguinis 9,644. Lalezari, P. and G.E. Bernard, 1966, J. Clin. Invest. 45, 1741. Lalezari, P., M. Nussbaum, S. Gelman and T.H. Spaet, 1960, Blood 15,236. Lalezari, P. and T.H. Spaet, 1959, Federation Proc. 18, 85. Lalezari, P. and T.H. Spaet, 1961, J. Lab. Clin. Meal. 57,868. Lalezari, P., W.J., Weinstein and B. Thalenfeld, 1970, in Terasaki, P.I. (ed.): Histocompatibilit} Testing, Munksgaard, Copenhagen, p. 319. Moeschlin, S. and K. Wagner, 1952, Schweiz. Med. Wochenschr. 82, 1104. Wasastjerna, C., Acta Med. Scan. 149,355.